1. Field of the Art
This invention relates to the use of a novel L-glutamic acid oxidase, particularly to the use thereof for analysis of L-glutamic acid.
More specifically, the present invention relates to an analytical method for assay of L-glutamic acid in a sample to be analyzed by the use of a novel L-glutamic acid oxidase which exhibits a strong affinity and a high substrate specificity for L-glutamic acid but has substantially no action on other amino acids and yet has a high stability, a reagent for analysis to practice the analytical method, a kit for analysis comprising the reagent, and a biosensor employing the enzyme.
2. Prior Art
As methods for analysis of L-glutamic acid, the chromatographic method, the microbiological quantitative determination method, the electrophoresis method, and the enzymatic method have been known. Among these methods, the chromatographic method and the enzymatic method are most generally used.
As the chromatographic method, the method in which an amino acid autoanalyzer is employed is generally practiced. This method, while it is an excellent method of high precision as well as high reliability, uses an expensive apparatus and may also involve a problem in that deproteinization may be sometimes required depending on the sample, thus making the method for sample preparation complicated.
The enzymatic methods known in the art are (1) the method in which an L-glutamic acid decarboxylase is used and (2) the method in which an L-glutamic acid dehydrogenase is used. However, the methods employing these enzymes have the following problems.
In the method (1) wherein an L-glutamic acid decarboxylase is employed, measurement of the amount of L-glutamic acid is carried out by detection of carbon dioxide which is the reaction product, for which there is generally employed (a) the method using a Warburg manometer or (b) the method using an autoanalyzer.
The method (a) using a Warburg manometer affords high precision, but it requires considerable skill of an expert, takes a long time for measurement, and also has a low sample-processing capacity. On the other hand, in the method (b) using an autoanalyzer, carbon dioxide is absorbed in a sodium carbonate solution of phenolphthalein, and the amount of carbon dioxide generated is measured by the degree of color reduction. Therefore preliminary treatments such as degassing of carbon dioxide and oxygen from an enzyme solution and a buffer are required under cooling, and also gas-liquid separation is necessary after the reaction, whereby the apparatus becomes disadvantageously complicated.
According to this enzymatic method, as an L-glutamic acid decarboxylase, an enzyme obtained from a pumpkin or E. coli is generally used. The enzyme from a pumpkin has a urease activity, and therefore when a sample containing urea is to be measured, measurement errors due to carbon dioxide from the urea may occur. The activity of this enzyme is also inhibited by an organic acid such as acetic acid, etc., and therefore, when a sample contains a large amount of organic acids, the enzymatic activity may be inhibited by the acids contained in the sample to provide inaccurate results. On the other hand, an enzyme from E. coli exhibits activities for L-arginine and L-glutamine, and therefore an accurate result cannot be obtained for a sample containing such a large amount of these amino acids as to have a substantial influence on the analysis of L-glutamic acid. Also, storage stability of the enzyme per se is not good.
The L-glutamic acid dehydrogenase used in the method (2) catalyzes the reaction in which L-glutamic acid is deaminated in the presence of NAD (oxidized form of nicotinamide adenine dinucleotide) to form .alpha.-ketoglutaric acid and ammonia accompanied with the formation of NADH (reduced form of nicotinamide adenine dinucleotide). In the method using this enzyme, measurement of the reaction is conducted by detection of the amount of NADH formed through the increase in absorbance at 340 nm. However, the equilibrium in this enzymatic reaction is more inclined toward formation of L-glutamic acid, and, for analysis of L-glutamic acid by the use of this enzyme, the equilibrium of this reaction must be shifted toward formation of .alpha.-ketoglutaric acid, and various contrivances are required therefor. For this purpose, a trapping agent for .alpha.-ketoglutaric acid is generally added to the reaction system, but such an agent may sometimes interfere with the reaction unless its concentration is strictly controlled. Further, the NAD concentration must also be controlled strictly. In the case of measuring a sample containing a substance exhibiting absorption at the wavelength to be measured, such as soy sauce, the value obtained must be corrected by using the value obtained in a blank test. Also, a lactic acid dehydrogenase may sometimes exist in the enzyme employed, and the influence of such an enzyme must also be taken into consideration.
Recently, an L-amino acid oxidase having a substrate specificity for L-glutamic acid has been found to be produced by cultivation of a microorganism belonging to the genus Streptomyces (hereinafter sometimes abbreviated as "S."), more specifically Streptomyces violascens (See Japanese Patent Laid-Open Publication No. 43685/1982). The physicochemical properties of the glutamic acid oxidase (hereinafter sometimes abbreviated as "known enzyme") as a protein have not yet been clarified, but the known enzyme is described to have enzymological properties as follows.
(1) Substrate specificity
When the velocity of enzymatic reaction for L-glutamic acid is given as 100, the known enzyme has a relative activity of 8.4 for L-glutamine and 6.8 for L-histidine, exhibiting substantially no activity for other amino acids.
(2) Optimum pH
pH 5-6
(3) pH stability
Stable in the range of pH 3.5-6.5 (37.degree. C., maintained for one hour)
(4) Temperature stability
Stable up to 50.degree. C. (maintained for 10 minutes) (5) Influence of inhibitors
Substantially completely inhibited by mercury ions, copper ions and diethyldithiocarbamate.
The specification of the above Laid-Open Publication states that a liquid culture of the aforesaid microorganism is preferable for production of the known enzyme.
For utilization of the known enzyme for analysis of L-glutamic acid, various problems are involved. Specifically, although the known enzyme has a higher substrate specificity for L-glutamic acid as compared with L-amino acid oxidases known in the art, it still exhibits clear activities for other amino acids as mentioned above, and therefore it cannot be used for specific quantitative determination of L-glutamic acid in the presence of these amino acids. Also, the known enzyme does not have a high pH stability and heat stability, and it cannot be considered to always have a good storage stability and stability during use as a reagent for analysis. Further, when copper ions exist in a sample to be analyzed, the activity of the known enzyme is markedly inhibited, whereby analysis may be considered to become difficult. Furthermore, the pH of reaction solutions employed in various clinical biochemical diagnostic analysis, especially in analysis of the activity of enzymes in blood, is usually around neutral, while the known enzyme will completely lose its activity at a pH of 7.5 when treated at 37.degree. C. for one hour. For this reason, it may be difficult to use the known enzyme in analysis around the neutral pH range.
The method for analysis of L-amino acid by the use of an L-amino acid oxidase has been known in the prior art, but it is difficult for known L-amino acid oxidases to act on L-glutamic acid, and therefore no specific analysis of L-glutamic acid has been possible according to the method in which such an enzyme is employed.
On the other hand, as another measure for analysis of L-glutamic acid, the method using a biosensor is known.
In the prior art, known biosensors for analysis of L-glutamic acid include (1) an enzyme electrode using L-glutamic acid dehydrogenase as the receptor portion of L-glutamic acid and a cation electrode as the transducer portion [Anal. Chim. Acta, 56, 333 (1971)] and (2) a microorganism electrode using the lyophilized cell of E. coli, which exhibits L-glutamic acid decarboxylase activity, as the receptor portion of L-glutamic acid and a carbon dioxide electrode as the transducer portion [Anal. Chim. Acta, 116, 61 (1980)].
The enzyme electrode of (1) is not practical since it has an extremely poor stability of the enzyme (only for 2 days) and also is susceptible to the influence of cations coexisting in the same measurement system such as sodium ion and potassium ion because of the use of a cation electrode. The microorganism electrode of (2) has an excellent stability (for 3 weeks, 1,500 times or more) because an immobilized microorganism cell is employed. However, carbon dioxide is generated through the aspiration action of the cell under aerobic conditions to exert an influence on the measurement. For removal of such an influence, it is necessary to inhibit the aspiration action of the cell and also to remove carbon dioxide contained in the sample by, for example, blowing nitrogen gas into the reaction mixture. Also, since the microorganism electrode does not have a high substrate specificity as tabulated below, it can be utilized only for rough measurements as in a process control of fermentation.
______________________________________ Substrate Specificity Microorganism Electrode Amino acid Relative sensitivity ratio ______________________________________ Glutamic acid 100 Glutamine 108 (11*) Alanine 0.5 Arginine 0.6 Aspartic acid 1.0 Cystine 0.4 Glycine 0.4 Tryptophan 0.4 ______________________________________ *Acetone treated cell was employed.
An enzyme electrode using an L-amino acid oxidase as the receptor portion is also known [Anal. Chem., 47, 1359 (1975)]. However, the L-amino acid oxidase of the prior art used in such an enzyme electrode acts on L-glutamic acid to a very small extent, and no specific analysis of L-glutamic acid has heretofore been possible by the use of the above enzyme electrode.
There is no disclosure in the above Laid-Open Publication as to whether or not the aforesaid known enzyme can be utilized for a biosensor for specific analysis of L-glutamic acid. Even if the known enzyme can be utilized as the receptor portion of a biosensor, it is possible for various problems to arise. That is, the known enzyme, while it has a relatively higher substrate specificity for L-glutamic acid as compared with L-amino acid oxidases known in the prior art, still exhibits clear activities for other amino acids as mentioned above, and therefore it cannot be utilized as the biosensor for specific analysis of L-glutamic acid in the co-presence of these amino acids. Also, the known enzyme does not have a high stability, and it cannot be considered to necessarily have a good storage stability and stability during use when utilized as the receptor portion of the biosensor. Further, since the known enzyme is extremely unstable in the pH range above 7 it may be difficult to use the known enzyme biosensor around the neutral pH range in which various biochemical analyses in the clinical field should be carried out. Furthermore, when copper ions are present in a sample to be analyzed, the activity of the known enzyme may be considered to be markedly inhibited by such ions, whereby analysis becomes difficult.
As described above, there has been in the prior art neither a biosensor for analysis of L-glutamic acid utilizing an oxidase as the receptor portion for specific recognition of L-glutamic acid nor an L-amino acid, oxidase capable of accomplishing sufficiently such an object.